IN GOLD: Dock control ligands against the 5RP site (use 5RP from2IYP aligned in PyMol to define the binding site) - Vicky Screen the libraries using this
Dock NADPH back into the NADPH site (use the NADPH from 2IYP aligned in PyMol to define the binding site) - Kavya -- use a LigPrepped version of NADPH from the PubChem site to be what you dock (should end up being 3 versions of NADPH after LigPrep) Determine which NADPH conformation looks best. Use the NAPDH + Tb6PGD as your receptor for a docking job of control ligands into the 5RP site (use 5RP from2IYP aligned in PyMol to define the binding site) Screen the libraries using this
Etiologic Risk Group (see link below):Appendix B-II-C Risk Group 2 (RG2)- Parasitic Agents *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Trypanosoma brucei is a protozoan that is known to cause African Sleeping sickness in humans. It originally is a endoparasite within the midgut of the tsetse fly, until it migrates to the salivary gland of the fly to later be injected into a mammalian host upon biting. The protist is then an endoparasite in the mammalian bloodstream. African Sleeping sickness is endemic to the subsaharan regions of Africa. The infection is curable with proper medication, but left untreated, it is commonly fatal. The course of the disease have two phases, differentiated by wether the parasite has crossed the blood-brain barrier and has thus infected the central nervous system. In the first phase, the parasite multiples in the tissues, blood, and lymph, and the infected individual suffers from fever, headaches, joint pain, and itching. In the second phase, the neurological phase, changes in behavior, sensory disturbances, poor coordination, confusion, and disruptions of the sleep cycle are common. Treatment of the disease is dependent upon how early the infection is detected and diagnosed. Continued research on the protozoan and the disease is necessary to improve the treatment options of those infected, improve the detection methods to limit the spread, and to develop a vaccination for those living in or traveling to endemic areas.
β-Nicotinamide adenine dinucleotide phosphate sodium salt hydrate (N-0505) $79.70 for 100mg
Glycylglycine Buffer= $77 per 25g
6-Phosphogluconate= $108 per 25un
ß-Nicotinamide Adenine Dinucleotide Phosphate Solution= $473 per kit (30 assays)
Magnesium Chloride= $43.60 per 100g
SUBSTRATE INFO NADP+
b-NICOTINAMIDE ADENINE DINUCLEOTIDE
PHOSPHATE
Sodium Salt
Product Number N0505 http://www.sigmaaldrich.com/catalog/product/sigma/n0505?lang=en®ion=US Preparation Instructionsβ-NADP+ is soluble in water at 50mg/ml.It is also soluble in methanol, much less soluble in ethanol andpractically insoluble in ether and ethyl acetate.Aqueous solutions stored as frozen aliquots are stable for at least one year.Repeated freeze thaw cycles are not recommended.Storage/StabilityStore at–20ºC Enzo Life Sciecnefor NADP+ http://www.enzolifesciences.com/ALX-480-003/nadp-.-disodium-salt/ soluble in water to 50mg/ml sesnsitive to alkaline - so store in water that is a little acidic.
Substrate Info (Dr. B) - 100113 May also dilute in Tris - talk to Kaarthik. NADPH - sigma N6505 (NOT THE RIGHT SUBSTRATE FOR THIS ENZYME UNLESS DOING THE BACK REACTION) http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/n6505pis.Par.0001.File.tmp/n6505pis.pdf Preparation Instructions b-NADPH is soluble in 0.01 M sodium hydroxide (50 mg/ml), yielding a clear, light yellow solution. Storage/Stability It is recommended to store Products N1630, N7505, and N6505 desiccated at –20 °C protected from light. Product N9910 can be stored at room temperature. The normal impurities and/or decomposition products are b-NADP and monophosphoadenosine 5¢- diphosphoribose. It is recommended to prepare solutions fresh and use promptly, unless you are sure this is an unnecessary precaution for your work. However, it has been reported that a 0.5 mM solution in 0.02 M NaOH (pH 12.3) showed no loss of purity in a week at 4 °C or -85 °C, but a 13% loss at –20 °C.3
For NADH and NADPH - the concern is that these are reduced and don't want them to be oxidized. So, want to reduce their exposure to oxygen - hence the NaOH.
Structure Available (PDB or Homology model)
-- PDB #: 1PGJ
Current Inhibitors: 4-phospho-D-erythronate and 2-deoxy-6-phosphono-D-gluconate are two of the current inhibitors with in vitro anti-parasitic activity.
Expression Information (has it been expressed in bacterial cells):Can be expressed in E.coli bacteria. http://www.sciencedirect.com/science/article/pii/S1046592884710060 Purification Method: 2-step purification mothod: DE-52 cellulose batch preparation followed by 2′ AMP-agarose affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
Figure 2: X-ray structure of 6-phosphogluconate dehydrogenase from the protozoan parasite T. brucei. *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
SMDVGVVGLGVMGANLALNIAEKGFKVAVFNRTYSKSEEFMKANASAPFAGNLKAFETMEAFAASLKKPRKALILVQAGA
ATDSTIEQLKKVFEKGDILVDTGNAHFKDQGRRAQQLEAAGLRFLGMGISGGEEGARKGPAFFPGGTLSVWEEIRPIVEA
AAAKADDGRPCVTMNGSGGAGSCVKMYHNSGEYAILQIWGEVFDILRAMGLNNDEVAAVLEDWKSKNFLKSYMLDISIAA
ARAKDKDGSYLTEHVMDRIGSKGTGLWSAQEALEIGVPAPSLNMAVVSRQFTMYKTERQANASNAPGITQSPGYTLKNKS
PSGPEIKQLYDSVCIAIISCYAQMFQCLREMDKVHNFGLNLPATIATFRAGCILQGYLLKPMTEAFEKNPNISNLMCAFQ
TEIRAGLQNYRDMVALITSKLEVSIPVLSASLNYVTAMFTPTLKYGQLVSLQRDVFGRHGYERVDKDGRESFQWPELQ *length of your protein in Amino Acids: 478 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 52.03 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 48360 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 3: TMpred graph image of 6-phosphogluconate dehydrogenase shows a few points above zero, but only 1 peak. This predicts 1 possible trans-membrane protein.
However, TDR targets predicted no trans-membrane proteins.
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) https://drive.google.com/?tab=mo&authuser=0#folders/0B4O2KqKh2q_-UnZrYzVIWlhRZVU
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): Forward Primer: 5’ TAC TTC CAA TCC ATG TCT ATG GAC GTT GGT GT 3’
Reverse Primer (complement): 5’ TAT CCA CCT TTA CTG TTA TTG CAG CTC CGG CCA CT 3’
*Target (protein/gene name): 6-phosphogluconate dehydrogenase
Virtual ToDo:
get control ligands
IN GOLD:
Dock control ligands against the 5RP site (use 5RP from2IYP aligned in PyMol to define the binding site) - Vicky
Screen the libraries using this
Dock NADPH back into the NADPH site (use the NADPH from 2IYP aligned in PyMol to define the binding site) - Kavya
-- use a LigPrepped version of NADPH from the PubChem site to be what you dock (should end up being 3 versions of NADPH after LigPrep)
Determine which NADPH conformation looks best.
Use the NAPDH + Tb6PGD as your receptor for a docking job of control ligands into the 5RP site (use 5RP from2IYP aligned in PyMol to define the binding site)
Screen the libraries using this
*NCBI Gene # or RefSeq#: 3660922
*Protein ID (NP or XP #) or Wolbachia#: XP_827463
*Organism (including strain): Trypanosoma brucei
TDR Targets:
http://tdrtargets.org/targets/view?gene_id=12199
Etiologic Risk Group (see link below): Appendix B-II-C Risk Group 2 (RG2)- Parasitic Agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Trypanosoma brucei is a protozoan that is known to cause African Sleeping sickness in humans. It originally is a endoparasite within the midgut of the tsetse fly, until it migrates to the salivary gland of the fly to later be injected into a mammalian host upon biting. The protist is then an endoparasite in the mammalian bloodstream. African Sleeping sickness is endemic to the subsaharan regions of Africa. The infection is curable with proper medication, but left untreated, it is commonly fatal. The course of the disease have two phases, differentiated by wether the parasite has crossed the blood-brain barrier and has thus infected the central nervous system. In the first phase, the parasite multiples in the tissues, blood, and lymph, and the infected individual suffers from fever, headaches, joint pain, and itching. In the second phase, the neurological phase, changes in behavior, sensory disturbances, poor coordination, confusion, and disruptions of the sleep cycle are common. Treatment of the disease is dependent upon how early the infection is detected and diagnosed. Continued research on the protozoan and the disease is necessary to improve the treatment options of those infected, improve the detection methods to limit the spread, and to develop a vaccination for those living in or traveling to endemic areas.
Essentiality of this protein: Essential. http://www.ncbi.nlm.nih.gov/pubmed/21363968
Complex of proteins?: No, but can be a dimer
Druggable Target: Yes http://www.ncbi.nlm.nih.gov/pubmed/21160204
*EC#: 1.1.1.44
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.44
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): reagents
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/6phosphoglucdehydro74.pdf
-- link (or citation) to paper that contains assay information
http://www.brenda-enzymes.info/literature/lit.php4?e=1.1.1.44&r=656470
-- List cost and quantity of substrate reagents and supplier
phosphogluconic acid SIGMA P7877 - $75 for 100mg
β-Nicotinamide adenine dinucleotide phosphate sodium salt hydrate (N-0505) $79.70 for 100mg
Glycylglycine Buffer= $77 per 25g
6-Phosphogluconate= $108 per 25un
ß-Nicotinamide Adenine Dinucleotide Phosphate Solution= $473 per kit (30 assays)
Magnesium Chloride= $43.60 per 100g
SUBSTRATE INFO
NADP+
b-NICOTINAMIDE ADENINE DINUCLEOTIDE
PHOSPHATE
Sodium Salt
Product Number N0505
http://www.sigmaaldrich.com/catalog/product/sigma/n0505?lang=en®ion=US
Preparation Instructionsβ-NADP+ is soluble in water at 50mg/ml.It is also soluble in methanol, much less soluble in ethanol andpractically insoluble in ether and ethyl acetate.Aqueous solutions stored as frozen aliquots are stable for at least one year.Repeated freeze thaw cycles are not recommended.Storage/StabilityStore at–20ºC
Enzo Life Sciecnefor NADP+
http://www.enzolifesciences.com/ALX-480-003/nadp-.-disodium-salt/
soluble in water to 50mg/ml
sesnsitive to alkaline - so store in water that is a little acidic.
6-phosphogluconate
P7877
http://www.sigmaaldrich.com/catalog/product/sigma/p7877?lang=en®ion=US
?? - no info on storage
Substrate Info (Dr. B) - 100113
May also dilute in Tris - talk to Kaarthik.
NADPH - sigma N6505 (NOT THE RIGHT SUBSTRATE FOR THIS ENZYME UNLESS DOING THE BACK REACTION)
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/n6505pis.Par.0001.File.tmp/n6505pis.pdf
Preparation Instructions
b-NADPH is soluble in 0.01 M sodium hydroxide
(50 mg/ml), yielding a clear, light yellow solution.
Storage/Stability
It is recommended to store Products N1630, N7505,
and N6505 desiccated at –20 °C protected from light.
Product N9910 can be stored at room temperature.
The normal impurities and/or decomposition products
are b-NADP and monophosphoadenosine 5¢-
diphosphoribose.
It is recommended to prepare solutions fresh and use
promptly, unless you are sure this is an unnecessary
precaution for your work. However, it has been
reported that a 0.5 mM solution in 0.02 M NaOH
(pH 12.3) showed no loss of purity in a week at 4 °C or
-85 °C, but a 13% loss at –20 °C.3
For NADH and NADPH - the concern is that these are reduced and don't want them to be oxidized. So, want to reduce their exposure to oxygen - hence the NaOH.
Structure Available (PDB or Homology model)
-- PDB #: 1PGJ
Current Inhibitors: 4-phospho-D-erythronate and 2-deoxy-6-phosphono-D-gluconate are two of the current inhibitors with in vitro anti-parasitic activity.
Expression Information (has it been expressed in bacterial cells):Can be expressed in E.coli bacteria.
http://www.sciencedirect.com/science/article/pii/S1046592884710060
Purification Method: 2-step purification mothod: DE-52 cellulose batch preparation followed by 2′ AMP-agarose affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
Figure 2: X-ray structure of 6-phosphogluconate dehydrogenase from the protozoan parasite T. brucei.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
SMDVGVVGLGVMGANLALNIAEKGFKVAVFNRTYSKSEEFMKANASAPFAGNLKAFETMEAFAASLKKPRKALILVQAGA
ATDSTIEQLKKVFEKGDILVDTGNAHFKDQGRRAQQLEAAGLRFLGMGISGGEEGARKGPAFFPGGTLSVWEEIRPIVEA
AAAKADDGRPCVTMNGSGGAGSCVKMYHNSGEYAILQIWGEVFDILRAMGLNNDEVAAVLEDWKSKNFLKSYMLDISIAA
ARAKDKDGSYLTEHVMDRIGSKGTGLWSAQEALEIGVPAPSLNMAVVSRQFTMYKTERQANASNAPGITQSPGYTLKNKS
PSGPEIKQLYDSVCIAIISCYAQMFQCLREMDKVHNFGLNLPATIATFRAGCILQGYLLKPMTEAFEKNPNISNLMCAFQ
TEIRAGLQNYRDMVALITSKLEVSIPVLSASLNYVTAMFTPTLKYGQLVSLQRDVFGRHGYERVDKDGRESFQWPELQ
*length of your protein in Amino Acids: 478
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 52.03 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 48360
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 3: TMpred graph image of 6-phosphogluconate dehydrogenase shows a few points above zero, but only 1 peak. This predicts 1 possible trans-membrane protein.
However, TDR targets predicted no trans-membrane proteins.
*CDS Gene Sequence (paste as text only):
ATGTCAATGGATGTCGGTGTTGTCGGCCTCGGGGTGATGGGCGCGAACCTGGCCTTGAACATTGCGGAGA AAGGGTTTAAAGTTGCTGTGTTCAACCGTACGTACTCTAAGAGCGAGGAATTCATGAAAGCGAATGCCTC TGCTCCATTTGCGGGTAATTTGAAGGCGTTTGAAACTATGGAGGCATTTGCAGCGTCACTCAAGAAACCT CGAAAGGCCCTCATCCTGGTGCAGGCGGGCGCGGCTACGGACTCAACAATTGAACAACTTAAGAAAGTGT TTGAGAAGGGAGATATCCTCGTTGATACCGGTAACGCGCATTTTAAGGATCAGGGACGCCGCGCCCAGCA GCTGGAGGCGGCAGGTCTCCGGTTTCTTGGGATGGGCATATCCGGGGGCGAGGAGGGTGCACGCAAAGGG CCAGCCTTTTTCCCTGGAGGGACGCTTAGTGTGTGGGAGGAAATACGACCAATTGTTGAGGCCGCCGCAG CTAAGGCAGATGATGGCCGGCCCTGTGTGACGATGAACGGCAGCGGCGGCGCGGGATCATGCGTGAAGAT GTACCACAATTCGGGTGAATACGCCATTTTGCAAATTTGGGGTGAGGTTTTTGACATCCTTCGGGCAATG GGACTGAACAACGATGAAGTTGCTGCCGTTCTTGAAGATTGGAAATCAAAGAACTTCTTGAAGTCTTATA TGCTCGATATCTCAATTGCAGCCGCGCGGGCAAAGGATAAGGATGGAAGTTATCTTACGGAGCACGTGAT GGATCGTATTGGATCGAAGGGCACCGGCTTATGGTCCGCCCAAGAGGCTCTCGAGATTGGAGTCCCTGCG CCCAGTTTGAACATGGCTGTCGTATCGCGGCAGTTCACAATGTATAAAACTGAGCGTCAAGCGAATGCCA GCAATGCACCCGGTATTACTCAATCCCCTGGATACACTCTCAAAAACAAAAGCCCCAGTGGGCCCGAAAT TAAGCAGCTCTACGACTCTGTGTGCATTGCCATTATCTCATGCTACGCTCAAATGTTTCAGTGCCTGCGT GAGATGGACAAGGTGCATAACTTCGGACTCAATCTTCCAGCTACCATTGCAACTTTCCGCGCCGGTTGCA TTTTGCAGGGCTACCTTTTAAAACCCATGACTGAGGCATTCGAAAAGAATCCCAACATTAGCAATCTCAT GTGTGCATTCCAAACCGAGATCAGGGCAGGACTACAGAATTACCGCGATATGGTGGCACTTATCACATCA AAGTTGGAAGTGTCCATTCCTGTGCTGTCGGCCTCCCTCAATTACGTTACTGCGATGTTTACGCCAACAC TCAAGTATGGGCAACTTGTGTCGTTGCAGCGGGATGTGTTCGGTCGGCACGGCTACGAAAGGGTGGATAA AGACGGCCGCGAATCATTCCAATGGCCTGAGTTGCAATAA
*GC% Content for gene: 51.39%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design
Protocol (paste as text only):
1 ATGTCTATGGACGTTGGTGTAGTTGGCCTGGGTGTTATGGGTGCGAACCTGGCGCTGAAT
61 ATTGCGGAAAAAGGTTTCAAAGTTGCGGTTTTCAACCGTACCTACTCTAAATCTGAAGAA
121 TTTATGAAAGCGAACGCTTCTGCACCGTTTGCGGGCAACCTGAAGGCGTTTGAGACCATG
181 GAAGCATTTGCGGCGTCTCTCAAGAAACCGCGTAAAGCGCTCATTCTGGTACAAGCGGGT
241 GCGGCCACCGACTCTACCATCGAACAGCTCAAAAAAGTATTTGAAAAGGGTGACATCCTG
301 GTCGACACCGGTAACGCGCATTTCAAGGACCAGGGTCGTCGTGCGCAGCAGCTGGAGGCT
361 GCGGGTCTCCGTTTCCTCGGCATGGGTATTTCTGGTGGTGAAGAAGGTGCGCGTAAGGGT
421 CCGGCGTTCTTCCCGGGTGGTACCCTGTCTGTATGGGAAGAGATTCGTCCGATCGTTGAA
481 GCTGCCGCTGCGAAAGCGGACGATGGTCGCCCGTGCGTAACGATGAATGGTTCCGGTGGT
541 GCTGGTTCTTGCGTTAAAATGTACCACAACAGCGGTGAATACGCGATCCTGCAAATCTGG
601 GGTGAAGTATTCGATATCCTGCGCGCGATGGGCCTGAATAATGACGAAGTAGCCGCGGTT
661 CTGGAAGACTGGAAAAGCAAAAACTTTCTCAAATCCTATATGCTGGACATCTCCATCGCT
721 GCGGCACGCGCTAAAGACAAGGACGGCTCCTACCTGACCGAGCACGTGATGGACCGTATC
781 GGTAGCAAAGGTACCGGTCTGTGGTCTGCCCAGGAAGCGCTGGAAATCGGTGTACCTGCG
841 CCATCCCTGAACATGGCGGTAGTTTCCCGCCAATTCACCATGTACAAGACCGAACGTCAG
901 GCGAATGCGTCTAACGCTCCGGGTATCACCCAGTCTCCTGGTTACACCCTGAAAAACAAA
961 TCTCCGTCTGGTCCGGAAATCAAACAACTGTACGACTCTGTTTGCATTGCGATCATCTCT
1021 TGCTACGCCCAAATGTTCCAATGCCTGCGTGAAATGGACAAAGTTCACAATTTCGGCCTC
1081 AACCTCCCAGCGACTATCGCGACCTTTCGTGCGGGCTGTATCCTCCAGGGTTACCTGCTG
1141 AAACCGATGACGGAGGCATTCGAGAAAAATCCGAACATCTCTAACCTCATGTGCGCATTC
1201 CAAACTGAGATCCGTGCCGGCCTCCAAAACTATCGTGATATGGTTGCTCTGATTACCTCT
1261 AAACTGGAAGTTTCTATCCCAGTTCTGAGCGCGAGCCTGAACTACGTAACCGCTATGTTC
1321 ACTCCGACGCTGAAATATGGCCAGCTCGTTTCTCTGCAGCGTGACGTTTTCGGTCGTCAT
1381 GGTTACGAACGTGTTGACAAAGATGGCCGCGAATCTTTTCAGTGGCCGGAGCTGCAATAA
1441
*GC% Content for gene (codon optimized): 49%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
https://drive.google.com/?tab=mo&authuser=0#folders/0B4O2KqKh2q_-UnZrYzVIWlhRZVU
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer:
5’ TAC TTC CAA TCC ATG TCT ATG GAC GTT GGT GT 3’
Reverse Primer (complement):
5’ TAT CCA CCT TTA CTG TTA TTG CAG CTC CGG CCA CT 3’
Resources:
Expression and Purification:http://www.sciencedirect.com/science/article/pii/S1046592884710060
Druggability: http://www.ncbi.nlm.nih.gov/pubmed/21160204
Essentiality: http://www.ncbi.nlm.nih.gov/pubmed/21363968
Brenda EC# http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.44
XP #: http://www.ncbi.nlm.nih.gov/protein/XP_827463.1
http://en.wikipedia.org/wiki/Trypanosoma_brucei
http://en.wikipedia.org/wiki/African_trypanosomiasis